Francisella tularensis is a highly infectious bacterium responsible for tularemia, a disease whose pneumonic form has potentially lethal consequences in humans. Francisella virulence depends on its ability to survive and replicate inside macrophages of the infected host. The current model of Francisella intracellular fate is initial enclosure within a phagosome, followed by escape from this phagosome and then replication in the cytoplasm, but the bacterial determinants controlling these individual stages are unknown. A Francisella pathogenicity island (FPI) has been identified as required for intracellular growth and likely encodes a Type 6 secretion system, but how it contributes to Francisella virulence is not understood. We have been using cell biology-, bacterial genetics- and genomics-based approaches to further characterize Francisella intracellular trafficking, identify genes expressed at various stages of the intracellular cycle and assess their role in Francisella virulence. [unreadable] [unreadable] In a model of murine primary macrophage infection with F. tularensis Type B and Type A strains, we have previously shown that phagosomal escape occurs rapidly after phagocytosis ( 20 min) using a fluorescence microscopy-based assay to measure phagosomal integrity (Checroun et al., 2006, PNAS, 103:14578). To understand what determines the efficiency of phagosomal disruption, we have further characterized the nature and maturation of the early Francisella-containing phagosome (FCP). Using advanced microscopy techniques, we have shown that early FCPs mature into an acidic vacuole prior to phagosomal disruption and that acidification is important for an efficient phagosomal escape. Furthermore, we have shown that the early FCP provides environmental cues for the induction of the FPI genes, which are required for phagosomal escape. Hence, Francisella senses intraphagosomal conditions generated by early FCP maturation events to express and activate its determinants of phagosomal disruption. This study is the subject of a manuscript under revision. We also have an ongoing collaboration with Dr Karl Klose (University of Texas at San Antonio) in which we use our expertise in the cell biology of Francisella-macrophage interactions to demonstrate the functionality of the FPI type 6 secretion system.[unreadable] [unreadable] In our efforts to identify Francisella genes that are important for intracellular pathogenesis, we have worked in collaboration with the RTB/RTS Genomics Unit at RML to establish the intracellular transcriptome of the Type A virulent F. tularensis subsp. tularensis Schu-S4 strain. The rationale for such an approach is that genes that are essential to intracellular survival and replication of Francisella should be upregulated inside macrophages, hence identifiable through transcriptional profiling of intracellular bacteria. We have optimized our procedures to isolate and amplify quality bacterial RNA from infected murine macrophages in sufficient amounts to obtain kinetic, transcriptional profiles of intracellular bacteria using the custom-made Affymetrix RML GeneChip II. The analysis of the microarray data has allowed us to characterize the genetic response of Francisella at the various stages of its intracellular cycle and revealed key aspects of its intracellular biology, including an efficient stress response during the endosomal stages and the upregulation of oligopeptide transport and amino acids catabolism during cytosolic replication. Known virulence determinants were upregulated during the infection cycle, therefore validating our approach. Additionally, several novel genes potentially involved in intracellular pathogenesis and/or virulence were identified, based on their expression profiles. To test this hypothesis, we have developed a method of allelic replacement in virulent Francisella and applied it to various virulence gene candidates. The generated mutants were examined for growth in macrophage and for virulence in mice, in collaboration with Katy Bosio. A mutant in the FTT0383 locus, which potentially encodes a transcriptional regulator, was highly attenuated in both macrophages and mice, confirming that our transcriptional approach successfully identified novel virulence determinants of Francisella. This study is the subject of a submitted manuscript.